Tissue engineered tendons and ligaments

ABSTRACT

Connective tissue, including neo-tendons and ligaments, has been constructed using biodegradable synthetic scaffolds seeded with tenocytes. The scaffolds are preferably formed from biodegradable fibers formed of a polymer such as polyglycolic acid-polylactic acid copolymers, and seeded with cells isolated from autologous tendon or ligament by means of enzymatic digestion or direct seeding into tissue culture dishes from explants. The cell polymer constructs are then surgically transplanted to replace missing segments of functioning tendon or ligament.

BACKGROUND OF THE INVENTION

The present invention is generally in the area of forming new tissues byimplantation of appropriate cells on a polymer matrix, and isspecifically directed towards construction of new tendons and ligaments.

Tissues connecting bones and muscles are collectively referred to hereinas “connective tissue”. Tendons are tissues which attach muscles tobones; aponeuroses are sheet-like tendons connecting one muscle withanother or with bones; ligaments hold bones together at joints. Tendonsand ligaments are elongated, cylindric structures formed of denseconnective tissue, adapted for tension in one direction, with fibershaving an orderly, parallel arrangement. The most common variety ofdense regularly arranged connective tissue has a predominance ofcollagenous (white) fibers arranged in bundles. Fibroblasts are placedin rows between the bundles. The tissue is silvery white, tough, yetsomewhat pliable. The collagen bundles of the tendons aggregate intolarger bundles that are enveloped by loose connective tissue containingblood vessels and nerves. Externally, the tendon is surrounded by asheath of dense connective tissue.

An essential characteristic of connective tissue is its strength andability to stretch or be pulled, then regain its original shape. Whendamaged, the orderly structure which imparts this ability to theconnective tissue is disrupted and usually does not heal to yield afully functional tissue.

Tendon defects, regardless of their origin, often prove to be difficultproblems for orthopedic surgery and hand surgery. An idealreconstruction repairs the defect with an autologous tendon graft.Autogenous tendon usage is limited by availability and donor sitemorbidity. Consequently, other approaches have been used: homo- orheterografts and artificial tendons. Homo- or heterografts, though,suffer from a scarcity of supply, increased susceptibility to infection,and concerns about possible transmission of infectious agents.

A carbon fiber implant for reconstructing severely torn ligaments andtendons has been developed which consists of carbon fibers coated with apolymer such as polylactic acid, as discussed in “Principles of HumanAnatomy” by G. J. Tortora, 5th edition (Harper & Row, NY 1989). Thecoated fibers are sewn in and around torn ligaments and tendons toreinforce them and to provide a scaffolding around which the body's owncollagenous fibers grow. The polymer is hydrolysed within the body overtime and the carbon fibers eventually fracture, typically within twoweeks. During this time, fibroblasts coat the fibers with collagen. Theoriginal structure of the tendons is not maintained, however, and therepaired tendon lacks strength and flexibility. Artificial prostheses,although not infectious, are also susceptible to infection, extrusion,and uncertain long-term immunologic interactions with the host.Moreover, prostheses cannot adapt to environmental stresses as do livingtendon tissue and have a high incidence of adhesive breakdown at theirinterface with the host.

It is therefore an object of the present invention to provide a methodand materials for creating new tendons and ligaments which have thestrength and flexibility of normal tendons and ligaments.

It is a further object the present invention to provide a method andmaterials for creating new tendons and ligaments which leaves no foreignmaterials in the body nor elicits an immunological reaction against thenew tendons or ligaments.

SUMMARY OF THE INVENTION

Connective tissue, including neo-tendons and ligaments, has beenconstructed using biodegradable synthetic scaffolds seeded withtenocytes. The scaffolds are preferably formed from biodegradable fibersformed of a polymer such as polyglycolic acid-polylactic acidcopolymers, and seeded with cells isolated from autologous tendon orligament by means of enzymatic digestion or direct seeding into tissueculture dishes from explants. The cell polymer constructs are thensurgically transplanted to replace missing segments of functioningtendon or ligament.

As shown by the examples, transplanted tenocytes attached tobiodegradable synthetic polymer scaffolds to generate new tendon inmice. Tenocytes were isolated from freshly slaughtered newborn calftendon and were seeded onto a non-woven mesh of polyglycolic acid,arranged as either a random array of fibers, or as fibers in parallel.The cell-polymer constructs were implanted into the mice subcutaneously.Specimens were harvested after six to ten weeks and examined. On grossexam nation, all specimens closely resembled tendons from which thecells had initially been isolated. Histologic evaluation demonstratedthat collagen bundles appeared to be organizing in a parallel fashion atthe Lateral aspects and appeared very similar to the collagen bundlesseen in normal tendon. Centrally, the collagen fibrils appeared to berandomly oriented. Specimens that were created from implantation ofparallel polymer fibers appeared to have a greater degree of parallelcollagen fibril orientation at an earlier time period. The neo-tendonconstructs demonstrated moderate tensile strength when stretched.

DETAILED DESCRIPTION OF THE INVENTION

A method and materials to form connective tissue, especially tendons andligaments, is described wherein cells obtained from tendons (tenocytes)or ligaments (ligamentum cells) are seeded onto and into biodegradable,biocompatible synthetic polymeric fibers, then implanted to form thedesired connective tissue.

Cells for Implantation

A variety of cells can be used to form connective tissue. Tenocytes andligamentum cells are the preferred cells. Fibroblasts differentiate toform collagen and can also be used. Dermal fibroblasts are preferred.Chondrocytes form collagen and can therefore be used, but are not aspreferred.

Autologous cells obtained by a biopsy are most preferred. Cells areisolated from autologous tendon or ligament by excision of tissue, theneither enzymatic digestion of cells to yield dissociated cells ormincing of tissue to form explants which are grown in cell culture toyield cells for seeding onto matrices. To obtain cells, the area to bebiopsied can be locally anesthetized with a small amount of lidocaineinjected subcutaneously. Alternatively, a small patch of lidocaine jellycan be applied over the area to be biopsied and left in place for aperiod of 5 to 20 minutes, prior to obtaining biopsy specimen. Thebiopsy can be obtained with the use of a biopsy needle, a rapid actionneedle which makes the procedure extremely simple and almost painless.This small biopsy core of tissue can then be transferred to mediaconsisting of phosphate buffered saline, divided into very small pieceswhich are adhered to a culture plate, and serum containing media added.Cells are dissociated as described below in the examples using standardtechniques, such as treatment with collagenase or trypsin.Alternatively, the tissue biopsy can be minced and the cells dispersedin a culture plate with any of the routinely used medias. After cellexpansion within the culture plate, the cells can be passaged utilizingthe usual technique until an adequate number of cells is achieved.

They can be maintained and/or proliferated in culture until implanted,either in standard cell culture dishes or after seeding onto matrices,as described below. Alternatively, cells can be seeded into and onto thematrix at the time of implantation.

Polymeric Matrices

Matrix Configuration

For a tendon or ligament to be constructed, successfully implanted, andfunction, the matrices must have sufficient surface area and exposure tonutrients such that cellular growth and differentiation can occurfollowing implantation. The organization of the tissue may be regulatedby the microstructure of the matrix. Specific pore sizes and structuresmay be utilized to control the pattern and extent of fibrovasculartissue ingrowth from the host, as well as the organization of theimplanted cells. The surface geometry and chemistry of the matrix may beregulated to control the adhesion, organization, and function ofimplanted cells or host cells.

In the preferred embodiment, the matrix is formed of polymers having afibrous structure which has sufficient interstitial spacing to allow forfree diffusion of nutrients and gases to cells attached to the matrixsurface until vascularization and engraftment of new tissue occurs.During this period of time, the implanted cells secrete new matrix whichincludes a parallel arrangement of type 1 collagen fibers as the polymersupport scaffolding degrades. The interstitial spacing is typically inthe range of 50 to 300 microns. As used herein, “fibrous” includes oneor more fibers that is entwined with itself, multiple fibers in a wovenor non-woven mesh, and sponge like devices.

Polymers

The matrices are formed of synthetic, biodegradable, biocompatiblepolymers. The term “bioerodible”, or “biodegradable”, as used hereinrefers to materials which are enzymatically or chemically degraded invivo into simpler chemical species. “Biocompatible” refers to materialswhich do not elicit a strong immunological reaction against the materialnor are toxic, and which degrade into non-toxic, non-immunogenicchemical species which are removed from the body by excretion ormetabolism.

In addition to biocompatibility, key characteristics of the polymer arethat it must be processable into fibers of an appropriate length,thickness, and strength for use as a matrix that serves to form newtendons or ligaments and it must degrade within the desired time frame,preferably six to twelve weeks, but optionally up to a few months oreven a year.

Fibers can be formed by melt-spinning, extrusion, casting, or othertechniques well known in the polymer processing area. Preferredsolvents, if used, are those which are completely removed by theprocessing or which are biocompatible in the amounts remaining afterprocessing.

Examples of polymers which can be used include natural and syntheticpolymers, although synthetic polymers are preferred for reproducibilityand controlled release kinetics. Synthetic polymers that can be usedinclude bioerodible polymers such as poly(lactide) (PLA), poly(glycolicacid) (PGA), poly(lactide-co-glycolide) (PLGA), and otherpolyhydroxyacids, poly(caprolactone), polycarbonates, polyamides,polyanhydrides, polyamino acids, polyortho esters, polyacetals,degradable polycyanoacrylates and degradable polyurethanes. Examples ofnatural polymers include proteins such as albumin, collagen, fibrin, andsynthetic polyamino acids, and polysaccharides such as alginate,heparin, and other naturally occurring biodegradable polymers of sugarunits.

PLA, PGA and PLA/PGA copolymers are particularly useful for forming thebiodegradable matrices. PLA polymers are usually prepared from thecyclic esters of lactic acids. Both L(+) and D(−) forms of lactic acidcan be used to prepare the PLA polymers, as well as the opticallyinactive DL-lactic acid mixture of D(−) and L(+) lactic acids. Methodsof preparing polylactides are well documented in the patent literature.The following U.S. Patents, the teachings of which are herebyincorporated by reference, describe in detail suitable polylactides,their properties and their preparation: U.S. Pat. No. 1,995,970 toDorough; U.S. Pat. No. 2,703,316 to Schneider; U.S. Pat. No. 2,758,987to Salzberg; U.S. Pat. No. 2,951,828 to Zeile; U.S. Pat. No. 2,676,945to Higgins; and U.S. Pat. Nos. 2,683,136; 3,531,561 to Trehu.

PGA is the homopolymer of glycolic acid (hydroxyacetic acid). In theconversion of glycolic acid to poly(glycolic acid), glycolic acid isinitially reacted with itself to form the cyclic ester glycolide, whichin the presence of heat and a catalyst is converted to a high molecularweight linear-chain polymer. PGA polymers and their properties aredescribed in more detail in Cyanamid Research Develops World's FirstSynthetic Absorbable Suture”, Chemistry and Industry, 905 (1970).

The erosion of the matrix is related to the molecular weights of PLA,PGA or PLA/PGA. The higher molecular weights, weight average molecularweights of 90,000 or higher, result in polymer matrices which retaintheir structural integrity for longer periods of time; while lowermolecular weights, weight average molecular weights of 30,000 or less,result in both slower release and shorter matrix lives. A preferredmaterial is poly(lactide-co-glycolide) (50:50), which degrades in aboutsix weeks following implantation (between one and two months).

All polymers for use in the matrix must meet the mechanical andbiochemical parameters necessary to provide adequate support for thecells with subsequent growth and proliferation. The polymers can becharacterized with respect to mechanical properties such as tensilestrength using an Instron tester, for polymer molecular weight by gelpermeation. chromatography (GPC), class transition temperature bydifferential scanning calorimetry (DSC) and bond structure by infrared(IR) spectroscopy, with respect to toxicology by initial screening testsinvolving Ames assays and in vitro teratogenicity assays, andimplantation studies in animals for immunogenicity, inflammation,release and degradation studies.

Polymer Coatings

In some embodiments, attachment of the cells to the polymer is enhancedby coating the polymers with compounds such as basement membranecomponents, agar, agarose, gelatin, cum arabic, collagens types I, II,III, IV, and V, fibronectin, laminin, glycosaminoglycans, polyvinylalcohol, mixtures thereof, and other hydrophilic and peptide attachmentmaterials known to those skilled in the art of cell culture. A preferredmaterial for coating the polymeric matrix is polyvinyl alcohol orcollagen.

Additives to Polymer Matrices

In some embodiments it may be desirable to add bioactive molecules tothe cells. A variety of bioactive molecules can be delivered using thematrices described herein. These are referred to generically herein as“factors” or “bioactive factors”.

In the preferred embodiment, the bioactive factors are growth factors.Examples of growth factors include heparin binding growth factor (hbgf),transforming growth factor a pha or Beta (TGFβ) , alpha fibroblasticgrowth factor (FGF), epidermal growth factor (TGF), vascular endotheliumgrowth factor (VEGF), some of which are also angiogenic factors. In someembodiments it may be desirable to incorporate factors such as nervegrowth factor (NGF) or muscle morphogenic factor (MMP). Steroidalantiinflammatories can be used to decrease inflammation to the implantedmatrix, thereby decreasing the amount of fibroblast tissue growing intothe matrix.

These factors are known to those skilled in the art and are availablecommercially or described in the literature. In vivo dosages arecalculated based on in vitro release studies in cell culture; aneffective dosage is that dosage which increases cell proliferation orsurvival as compared with controls, as described in more detail in thefollowing examples. Preferably, the bioactive factors are incorporatedto between one and 30% by weight, although the factors can beincorporated to a weight percentage between 0.01 and 30% weightpercentage.

Bioactive molecules can be incorporated into the matrix and releasedover time by diffusion and/or degradation of the matrix, or they can besuspended with the cell suspension.

Implantation

The matrices are implanted in the same manner as other reconstructed orprosthetic tendons or ligaments. They are surgically interposed betweenthe cut ends of autologous tendon and sutured in place withbiodegradable suture material.

The tendons and ligaments and other connective tissue are useful forrepair and reconstruction of congenital defects as well as traumaticdefects. The neo-connective tissue is therefore useful in plasticsurgery for cosmetic purposes as well as in reconstructive surgery.

The present invention will be further understood by reference to thefollowing non-limiting examples evaluating the feasibility oftransplanted tenocytes attached to biodegradable synthetic polymerscaffolds to generate new tendon in athymic mice. Tenocytes wereisolated from freshly slaughtered newborn calf tendon and were seededonto a non-woven mesh of polyglycolic acid, arranged as either a randomarray of fibers, or as fibers in parallel. The cell-polymer constructswere implanted into athymic mice subcutaneously. Specimens wereharvested after 6-10 weeks and examined. On gross examination, allspecimens (n=25) closely resembled tendons from which the cells hadinitially been isolated after 6 weeks on in vivo incubation. Histologicevaluation using a standard hematoxylin and eosin stain demonstratedpolymer remnants of cells embedded within collagen fibrils. The collagenbundle appeared to be organizing in a parallel fashion at the lateralaspects and appeared very similar to the collagen bundle seen in normaltendon. Centrally, the collagen fibrils appeared to be randomlyoriented. Specimens that were created from implantation of parallelpolymer fibers appeared to have a greater degree of parallel collagenfibril orientation at an earlier time period. The neo-tendon constructsdemonstrated moderate tensile strength when stretched.

EXAMPLE 1 Construction of Neo-tendon Method and Materials

Sheets approximately 100 microns thick composed of an embossed non-wovenmesh of polyglycolic acid with interfiber interstitial spacing averaging75 to 100 microns in diameter, arranged as either a random array offibers, or as fibers in parallel (Dexon, Davis and Geck), were cut intopieces approximately 0.4 cm×4 cm and set aside. Tendon was obtained fromthe shoulder of newborn calves within six hours of sacrifice. Thetendons were diced into pieces approximately 0.5 cm×0.5 cm and placed ina sterile 50 ml conical tube. The tendon pieces were washed twice withDulbecco's phosphate buffered saline (PBS) (Gibco, Grand Island, N.Y.).A sterile 0.396 Collagenase solution was prepared by mixing 75 mg oftype collagenase (Worthington, Freehold, N.J.) with 25 ml of Hamm's F-12medium (Gibco, Grand Island, N.Y.). The tendon fragments were incubatedin the collagenase solution for 12-16 hours at 37° C. on a shaker. Afterdigestion, the solution was filtered through a sterile 150 micron nylonmesh (Tetko, Elmsford, N.Y.) to remove undigested fragments, and thetenocytes were washed twice in 25 ml of PBS. Cells were counted using ahemocytometer and concentrated in a cell suspension containing 150×10tenocytes/ml. One hundred microliters of suspension (15 million cells)were then seeded onto each of 25 polymer constructs. Cell-polymerconstructs were placed into 35 mm tissue dishes with 4 ml of Hamm's F-12(Tissue Culture Media) with 1096 fetal calf serum (Gibco, Grand Island,N.Y.) with L-glutamine (292 μg/ml), penicillin (100 u/ml), streptomycin(100 μg/ml) and ascorbic acid (50 μg/ml) and kept in an incubator invitro at 37° C. in the presence of 596 CO₂ for one week until the fiberswere coated with multiple layers of tenocytes. Then, under generalanesthesia, 25 cell-polymer constructs were surgically implantedsubcutaneously into each of 25 nude mice (Athymic, NCr/nude/Sde, Dept.of Radiation Medicine at the Massachusetts General Hospital) four tofive weeks of age (the experimental group). An additional 10 micereceived implants of polymers containing no cells (the control group).Specimens were harvested after six to ten weeks of in vivo incubationand examined grossly and histologically for the evidence of tendonformation. The tensile mechanical property and handling characteristicsof each specimen was assessed.

Results

After six weeks, gross examination of all experimental specimens (n=25)closely resembled normal calf tendons from which the cells had beenisolated. Histologic evaluation with hematoxylin and eosin and Masson'sTrichrome staining demonstrates organized collagen fibrils with polymerremnants. The peripheral areas demonstrated a parallel linearorganization of longitudinal collagen fibrils similar to the collagenbundles seen in normal calf tendon. Centrally, however, the collagenfibrils in this early specimens were randomly oriented and lacked theparallel linear organization. At 10 weeks, histological evaluation showsparallel linear organization of collagen bundles throughout thespecimens, centrally and peripherally. Specimens created fromimplantation of tenocytes onto polymer fibers arranged in parallelfashion showed a greater degree of parallel collagen fibril organizationat six weeks when compared to specimens created from randomly arrangedpolymer fibers, control specimens (n=10), without tenocyte implantationshowed no evidence of tendon on gross or histological evaluation at tenweeks. Mechanical analysis of neo-tendon constructs have comparabletensile strength and similar mechanical characteristics to normaltendon. Tensile measurements showed that the tissue engineered tendonswere very similar in mechanical behavior to that of normal tendons. Theaverage tensile strength of the tissue engineered tendon after eightweeks in vivo was 10.75±2.29 MPa/normal tendon: 32.80±5.21 MPa standarddeviation of that of the normal tendons. The mechanical loading was duesolely to the new tendons formed, as the polymer scaffolds degrade andloss mechanical strength after four weeks.

The results demonstrate that tenocytes will adhere to syntheticbiodegradable polymers survive and multiply in vitro, and thattenocyte-polymer construct implantation in vivo results in formation oftendon with characteristics similar to normal mature tendon. Thecollagen fibrils undergo organization over time. Histological evaluationof the neo-tendon construct at six weeks shows linear organization, likenormal tendon, peripherally and random array of collagen fibrilscentrally. With parallel arranged polymer fibers, linear orientation ofcollagen centrally was achieved at an earlier time. At ten weeks, thecollagen fibers were arranged in parallel linear fashion throughout theshowed proper anatomic cellular organization regardless of polymer fiberorientation. Thus, parallel structural fibers facilitates earlyorganization of collagen, but the ultimate architectural organization isrelated to cellular communication and interaction and not of polymerorientation.

Variations and modifications of the present invention will be obviousfrom the foregoing detailed description of the invention. Suchmodifications and variations are intended to come within the scope ofthe following claims.

1. A connective tissue construct comprising synthetic, biodegradablebiocompatible polymer fibers with an interstitial spacing betweenapproximately 50 and 300 microns forming a matrix suitable forimplantation to form connective tissue having dissociated connectivetissue cells therein in an amount effective to form connective tissuefollowing implantation into a patient in need thereof.
 2. The constructof claim 1 wherein the cells are selected from the group consisting oftenocytes, ligamentum cells, fibroblasts, and chondrocytes.
 3. Theconstruct of claim 1 wherein the polymer is selected from the groupconsisting of poly(lactide), poly(glycolic acid),poly(lactide-co-glycolide), poly(caprolactone), polycarbonates,polyamides, polyanhydrides, polyamino acids, polyortho esters,polyacetals, degradable polycyanoacrylates, degradable polyurethanes,proteins, and polysaccharides.
 4. The construct of claim 1 wherein theconstruct is implanted to form new tendon.
 5. The construct of claim 1wherein the construct is implanted to form new ligament.